1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Role of microbes in the management of cassava mosaic disease(Department of molecular biology and biotechnology, college of agriculture , Vellayani, 2023-09-18) Athulya, V AThe study entitled “Role of microbes in the management of Cassava Mosaic Disease” was carried out at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute and the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani in the year 2022-2023 to analyse the role of microbial biocontrol agents in reducing the incidence of Cassava Mosaic Disease. Infected cuttings of two susceptible cassava varieties H226 and Sree Prakash were collected from the fields at ICAR-CTCRI. They were identified to be infected with Cassava Mosaic Virus by visual observation of disease symptoms. DNA was isolated from the leaves of these plants by CTAB method and PCR was performed to confirm infection using the coat protein primer pair. Ten isolates of Trichoderma asperellum and Bacillus sp. were used. They were cultured in Potato Dextrose Broth and Nutrient Broth respectively. The infected cuttings were immersed in the microbial suspensions and treated using a sett treating device for 15 min at 15 lbs pressure. They were then planted in trays in a mixture of coir pith and soil, along with untreated control. Six replicates were used for all. Biometric observations like number of leaves, branch length and symptom scores were recorded for three months from the date of planting. DNA was isolated at one and three months after planting and PCR was performed to confirm infection. qPCR was performed on this DNA to check the viral load in the plants. Three isolates of Trichoderma asperellum (T4, T7 and T8) showed reduction in viral load as compared to control in H226 plants. After the first month, plants treated with T4 had a viral load of 1.36x108 copies/10ng and T7 had 1.26x105 copies/10ng while control had 105 copies/10ng. After three months, plants treated with T4 had 53.2 copies/10ng, T7 had 58.5 copies/10ng and T8 had 68.5 copies/10ng while control had 3.9x109 11 2 copies/10ng. Two isolates (B3 and B8) of Bacillus sp. showed reduction in viral load in Sree Prakash variety. One month after planting, plants treated with B3 (Bacillus subtilis) had a viral load of 1.27x109 copies/10ng while untreated control had 7.4x105 copies/10ng. After three months, plants treated with B3 had a viral load of 1.74x104 copies/10ng and those treated with B8 had 8.1x103 copies/10ng while control had 8.7x105 copies/10ng. The biometric observation did not always show correlating trends. In Trichoderma treated H226 plants, the percentage changes in disease severity were 9.1% reduction for T7 and 10.9% reduction for T8 over control after one month. After three months, there was a 25% increase for T4, 8.3% increase for T7 and 4.2% increase for T8 in disease severity over control. In Sree Prakash plants treated with Bacillus sp., the percentage change in disease severity was 24% increase for both treatments B3 and B8 after one month, over control. After three months, the percentage change in disease severity was 26.75% increase with B3 over control. Primers were designed for two plant defense genes ETR1 and PAL1 using Primer3 online tool for future study of the expression of these genes. This may help understand the mechanism of action of these biocontrol agents in reducing viral load. Thus, certain isolates of Trichoderma asperellum and Bacillus sp. were found to have effective biocontrol activity against Cassava Mosaic Disease in this preliminary study. Further studies with a larger number of replicates and longer growth period is necessary for proper validation of the results.Item Role of microbes in the management of cassava mosaic disease(Department of molecular biology and biotechnology, college of agriculture, Vellayani, 2023-09-18) Athulya, V A.; Makeshkumar, TThe study entitled “Role of microbes in the management of Cassava Mosaic Disease” was carried out at the Division of Crop Protection, ICAR- Central Tuber Crops Research Institute and the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani in the year 2022-2023 to analyse the role of microbial biocontrol agents in reducing the incidence of Cassava Mosaic Disease. Infected cuttings of two susceptible cassava varieties H226 and Sree Prakash were collected from the fields at ICAR-CTCRI. They were identified to be infected with Cassava Mosaic Virus by visual observation of disease symptoms. DNA was isolated from the leaves of these plants by CTAB method and PCR was performed to confirm infection using the coat protein primer pair. Ten isolates of Trichoderma asperellum and Bacillus sp. were used. They were cultured in Potato Dextrose Broth and Nutrient Broth respectively. The infected cuttings were immersed in the microbial suspensions and treated using a sett treating device for 15 min at 15 lbs pressure. They were then planted in trays in a mixture of coir pith and soil, along with untreated control. Six replicates were used for all. Biometric observations like number of leaves, branch length and symptom scores were recorded for three months from the date of planting. DNA was isolated at one and three months after planting and PCR was performed to confirm infection. qPCR was performed on this DNA to check the viral load in the plants. Three isolates of Trichoderma asperellum (T4, T7 and T8) showed reduction in viral load as compared to control in H226 plants. After the first month, plants treated with T4 had a viral load of 1.36x108 copies/10ng and T7 had 1.26x105 copies/10ng while control had 105 copies/10ng. After three months, plants treated with T4 had 53.2 copies/10ng, T7 had 58.5 copies/10ng and T8 had 68.5 copies/10ng while control had 3.9x109 11 2 copies/10ng. Two isolates (B3 and B8) of Bacillus sp. showed reduction in viral load in Sree Prakash variety. One month after planting, plants treated with B3 (Bacillus subtilis) had a viral load of 1.27x109 copies/10ng while untreated control had 7.4x105 copies/10ng. After three months, plants treated with B3 had a viral load of 1.74x104 copies/10ng and those treated with B8 had 8.1x103 copies/10ng while control had 8.7x105 copies/10ng. The biometric observation did not always show correlating trends. In Trichoderma treated H226 plants, the percentage changes in disease severity were 9.1% reduction for T7 and 10.9% reduction for T8 over control after one month. After three months, there was a 25% increase for T4, 8.3% increase for T7 and 4.2% increase for T8 in disease severity over control. In Sree Prakash plants treated with Bacillus sp., the percentage change in disease severity was 24% increase for both treatments B3 and B8 after one month, over control. After three months, the percentage change in disease severity was 26.75% increase with B3 over control. Primers were designed for two plant defense genes ETR1 and PAL1 using Primer3 online tool for future study of the expression of these genes. This may help understand the mechanism of action of these biocontrol agents in reducing viral load. Thus, certain isolates of Trichoderma asperellum and Bacillus sp. were found to have effective biocontrol activity against Cassava Mosaic Disease in this preliminary study. Further studies with a larger number of replicates and longer growth period is necessary for proper validation of the results.Item Zinc homeostasis and carbon sequestration as influenced by biofertilizers and CO2 enrichment in rice (Oryza sativa L.)(Department of plant pathology, college of agriculture ,Vellayani, 2023-07-21) Amrutha, E A.; Manju, R VThe study entitled "Zinc homeostasis and carbon sequestration as influenced by biofertilizers and CO2 enrichment in rice (Oryza sativa L.)" was undertaken with the objectives of assessing the impact of biofertilizers on Zn uptake, translocation and soil carbon sequestration and the mechanism of Zn transport as influenced by CO2 enrichment. The first experiment was conducted to study the influence of biofertilizers on Zn uptake, translocation, and carbon sequestration at IFSRS, Karamana. The experiment was laid out in RBD with eleven treatments and three replications. The treatments comprised of T1: PoP, KAU + Azolla, T2: PoP, KAU + AMF, T3: PoP, KAU + PGPR Mix I, T4: PoP, KAU + KSB, T5: PoP, KAU + PSB, T6: T1 + AMF, T7: T1 + PGPR Mix I, T8: T1 + KSB, T9: T1 + PSB, T10: Control and T11: PoP+ Zinc sulphate spray. The gene expression analysis revealed the down regulation of OsZIP4 genes under elevated CO2 compared to ambient CO2. Biofertilizer induced overexpression of OsZIP 4 genes is found to play significant role in improving Zn translocation. The study results could be helpful to formulate climate smart agricultural practices strategies to address the wide spread problem of limited availability of Zn among the populationItem Bioecology of major coccinellid predators of Kerala(Department of agricultural entomology, college of agriculture , Vellayani, 2023-07-07) Anusree, S S; Anitha, NAn investigation on “Bioecology of major coccinellid predators of Kerala” was carried out at Department of Entomology, College of Agriculture, Vellayani during 2017-2022 with the objective to identify major coccinellid predators of pests infesting agricultural crops from agro ecological zones of Kerala and to study biology and predatory potential of select coccinellids. In the present investigation, 40 species of predatory coccinellids belonging to 23 genera under 6 tribes in the Subfamily Coccinellinae were illustrated. Taxonomic study on tribes Aspidimerini and Chilocorini resulted in the identification of three species in each tribe. Examination on specimens of tribe Coccidulini resulted in identifying 16 species within five genera. 11 species belonging to nine genera were illustrated under tribe Coccinellini. A single species was illustrated and studied under tribe Hyperaspidini. The specimens studied under tribe Sticholotidini belonged to six species within four genera. Among the 40 illustrated species, 28 species were identified, while identity of 12 species are to be confirmed, of which three are putative new species. Phrynocaria perfida Poorani collected and illustrated during this investigation were confirmed and described as a new species (Poorani et al., 2021). Chilocorus sp.1 and Scymnus (Pullus) sp.4 are the other two putative new species. Phrynocaria perrotetti (Mulsant), Cryptogonus orbiculus (Gyllenhal) and Sticholotis ferruginea (Gorham) are new records from Kerala.Item Genetic diversity analysis of indigenous rice varieties in Kerala using molecular markers(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Ajith, M K; Jayalekshmy, V GThe present study entitled “Genetic diversity analysis of indigenous rice varieties in Kerala using molecular markers “was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Thiruvananthapuram during 2017-2018. The study was conducted with the objective to analyze the genetic diversity of traditional rice varieties in four agro climatic zones of Kerala using RAPD and SSR markers. Five varieties were collected from the each agro climatic zones viz., hill areas of Wayanad, rice growing tract of Palakkad, saline soils of Pokkali and Kuttanad soils. The DNA was isolated and RAPD analysis with 10 Operon primers and SSR analysis was done with ten RM primers. In the RAPD analysis the ten operon primers produced 88 amplicons with an average polymorphism 82%. Resolving power OPC-07 (15.4) had the highest value but its Polymorphism Information Content and Effective Multiplication Ratio (EMR) were considerably low. Considering all the three parameters together primer OPF-06 is found to be the best RAPD primer with considerably high polymorphism information content, resolving power and effective multiplication ratio. The dendrogram constructed based on the RAPD scoring showed that varieties Pokkali and VTL-2 had maximum similarity. These two were from Pokkali rice tract. PTB 12 from Pattambi was found to be unique and it clustered with others only at 30% similarity.The clustering of the genotypes did not show any correlation with the geographic origin. ABL 12 and VTL- 2 showed 70 % similarity but those two were from Wayanad Hills and Pokkali tract respectively. Vellakuttadan from Moncombu clustered with PTB 2 from pattambi at 72% a similarity. Kochuvith and Vellakuttadan from Moncombu clustered at 67 %. All the SSR markers produced two alleles except RM 210 and RM 204 which produced four alleles and one allele respectively. All the alleles of all the markers were polymorphic except that of primer RM204. The polymorphism information content of the SSR primers used in the study ranged from 0 to 0.88. In this study the highest PIC value of 0.88 was reported by RM 210 followed by RM 567 (0.85). The resolving power and EMR was also highest for RM 210. The dendrogram constructed based on the SSR markers could give a clustering of genotypes more correlated with the geographic origin. Genotypes Kochuvithu and Vellakuttadan showed 100 % similarity both where from Kuttanad. But Karavalakochuvith, T.virippu, and PTB 2 also showed 100% similarity but these three were from Moncompu, Pokkali and Palakkad respectively. At around 90 % similarity AMB 14, AMB 22 from Wayanad, Pokkali andVTL-2 from Pokkali, PTB 13 and PTB 8 from Palakkad clustered showing more correlation to the Geographic origin. SSR markers being sequence specific and flanking the repeat sequence which has more role in evolution, are more reliable in predicting the Genetic diversity based on origin. Since both of them could not give a clear-cut clustering based on geographic origin an analysis using both the markers together was done. This gave a better picture of the clustering as it involved more number of variables. But here also the varieties from Wayanad A1 to A5 were scattered in different clusters. Only A 14 and A15 (AMB 14 and AMB 5) clustered at 60 percentage similarity. The accessions from moncompu (A6-A10) clustered at around 50 % similarity. In the accession from Pokkali tract (A11-A15) only T.virippu to VTL-2 clustered at 78 % similarity. Accession from central zone Pattambi (A16-A20) was scattered into different clusters. PTB 12 was unique from other accessions. This molecular diversity analysis of the traditional rice genotypes from four different agro climatic zones could find that the maximum similarity was 78% and that too only between two accessions. The diversity among the genotypes was 64% as all the genotypes clustered at 36% similarity. The clustering of the genotypes did not show any correlation with the geographic origin. Exchange of varieties between the farmers and some amount of natural crossing would have led to the mixture of populations of rice genotypes in different agro climatic zones.Item Nutrient based management of chilli leaf curl virus in Chilli (Capsicum annuum L.)(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Shilpa Sankar; Radhika, N SThe study entitled “Nutrient based management of Chilli leaf curl virus in Chilli (Capsicum annuum L.) was conducted at Department of Plant Pathology, College of Agriculture, Vellayani from 2017 to 2019. The main objectives were to serologically and molecularly characterize the virus causing leaf curl in chilli and to study the role of nutrient application in the management of the disease. The present investigation was carried out in four experiments viz., collection of Begomovirus infecting chilli from different cultivated areas, symptomatology, serological diagnosis and molecular characterization of the virus causing chilli leaf curl and nutrient based management of chilli leaf curl. The survey was undertaken from December 2018 to March 2019 to investigate the disease incidence in major chilli growing areas of Palakkad (Vadakarapathy and Kozhinjanpara) and Thiruvananthapuram districts (College of Agriculture, Vellayani) of Kerala. In Thiruvananthapuram, the disease incidence ranged from 69.33 to 80 per cent whereas Palakkad recorded maximum incidence of 73.33 per cent in Vadakarapathy village and Kozhinjanpara village recorded the disease incidence of 55.71 to 71.70 per cent. The common symptoms of the disease were upward curling and puckering of leaves, and stunting of whole plant. Other symptoms viz., reduced leaf size, yellowing, petiole elongation, crinkling and mottling of leaves with few or no fruits were also observed. Serological diagnosis of the disease was carried out using triple antibody sandwich – enzyme linked immunosorbent assay (TAS-ELISA) using polyclonal antisera specific to Begomovirus, Sri Lankan cassava mosaic virus (SLCMV). All the samples collected from College of Agriculture, Vellayani were detected with the virus whereas none of the samples from Palakkad district were positive to the virus. Molecular characterization of the virus using universal primers specific to coat protein of Begomovirus viz., AV-AC and DENG through Polymerase chain reaction (PCR) revealed that the DNA isolated from samples of Vellayani could yield an amplicon size of ̴ 550 bp (AV-AC) and ̴ 500 bp (DENG); thus confirming the presence of the virus. But no PCR amplification was observed in any of the samples from Palakkad district. Blast analysis with the sequence of coat protein of Vellayani revealed that the virus had 96.63 per cent similarity with Chilli leaf curl Vellanad virus. Nutrient status of healthy and diseased leaves revealed that the per cent of major nutrients viz., nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg); and micronutrients viz., manganese (Mn), zinc (Zn), copper (Cu) and boron (B) in the infected leaves was low compared to the healthy leaves but in sufficient level. Whereas, the per cent of sulphur (S) and iron (Fe) in infected leaves was high and in toxic level. Comparatively higher levels of all the major and micro nutrients were recorded in the soils collected from the infected fields than the soils from the healthy field. A pot culture study was conducted at Department of Plant Pathology in completely randomized design consisting of ten treatments and three replications with chilli variety Vellayani Athulya from May 2019 to August 2019. Cent per cent disease incidence was recorded in all chilli plants before imposing the treatments. Soil samples were taken and analysed for major and micro nutrients at pre-treatment stage. Urea, rajphos, murate of potash, lime and magnesium sulphate (MgSO4) were used as the source of major nutrients like N, P, K, Ca and Mg, respectively. Whereas, manganese sulphate (MnSO4), zinc sulphate (ZnSO4), copper sulphate (CuSO4), borax and potassium silicate were used as source for micronutrients such as Mn, Zn, Cu, B and Si, respectively. After imposing the treatments, the highest coefficient of infection (75.0) was recorded in untreated infected plants whereas, the plants supplemented with nutrients as per package of practices (POP) + B @ 10 kg ha-1, POP + (ZnSO4) @ 20 kg ha-1 and, basal application of 1/2 N + full P + 1/2 K followed by 0.5 per cent foliar application of NPK 19:19:19 at fortnightly recorded the lowest coefficient of infection (25.0). TAS-ELISA conducted with all the experimental plants confirmed the presence of virus. Lower virus titre value was observed in the plants supplemented with nutrients as per POP recorded maximum level of major nutrients. Among the treatments highest fruit yield of 48.85 g plant1 was obtained from plants supplemented with nutrients as per POP + B @ 10 kg ha-1 was found to be most effective in reducing the coefficient of infection with better yield. Thus, the present study revealed that the serological diagnosis of the disease carried out using TAS-ELISA with antisera SLCMV and molecular characterization of the virus using primers AV-AC and DENG through PCR confirmed the presence of virus in Vellayani. The BLAST analysis of Chilli leaf curl Vellayani isolate thus showed 96.63 per cent similarity with Chilli leaf curl Vellanad virus. It was also indicated that Chilli leaf curl virus in chilli could be managed by the application of nutrients as per package of practices recommendation along with boron as borax @ 10 kg ha-1 which need to be validated under field conditions.Item Development of recombinant coat protien for immunodetection of cucumber mosaic virus infecting banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Alan C Antony; Vimi LouisBanana (Musa spp.) is infected by four well characterised plant viruses viz., Banana bunchy top virus, Cucumber mosaic virus (CMV), Banana bract mosaic virus and Banana streak virus. Among these, CMV causes devastating effect on tissue culture banana plants. The study entitled “Development of recombinant coat protein for immunodetection of Cucumber mosaic virus infecting banana” was carried out using existing facilities of Department of Biochemistry, Indian Institute of Science, Bangalore, Division of Plant Pathology, Banana Research Station, Kannara and Department of Plant Pathology, College of Horticulture, Vellanikkara, Thrissur during 2018- 2019. The present study was carried out to produce recombinant coat protein, which can be utilised later for producing high quality antiserum for the detection of CMV infecting banana. Cucumber mosaic virus infected samples were collected based on various characteristic symptoms and screened by direct antigen coating immunosorbent assay using commercially available CMV polyclonal antiserum. Isolate namely, KANC- 4, KANC- 2 and NDRNS- 4 showed maximum absorbance at 405 nm and hence selected for molecular detection using reverse transcriptase polymerase chain reaction with CMV- CP specific primer. The PCR product was purified and CMV- CP amplicon of NDRNS- 4 isolate was ligated to pGEM- T linear plasmid vector, which was later transformed into E. coli DH5α cells. Positive clones were selected according to blue-white screening. Cloning i.e., E.coli DH5α/pGEM- T/CMV- CP was confirmed through colony PCR using coat protein specific primer, restriction digestion of recombinant plasmids using EcoR1 enzyme followed by sequencing. The vectors viz. pRSET- C and pET28a were selected for the expression of CMV- CP gene in E. coli. Coat protein specific forward (5’GGG GCT AGC ATG GAC AAA TCT GAA TCA ACC3’) and reverse primers(5’CCC GGA TCC TTA CTC TCC ATG GCG TTT AG 3’) were designed along with recognition sites of restriction enzymes BamH1 and Nhe1.The annealing temperature of designed primer was standardised as 55°C using gradient PCR. The coat protein gene of CMV was amplified at 750 bp using designed primers and high fidelity Pfu DNA polymerase enzyme. Expression vectors as well as amplicon were subjected to ligation and the recombination in expression plasmids (pRSET- C/ CMV- CP and pET28a/CMV- CP) were confirmed through PCR and sequencing. The plasmid with maximum homology i.e., pRSET-C/CMV- CP was selected for further studies. The recombinant plasmid was transformed into E. coli BL21(DE3)pLysS cells for the expression of CMV- CP gene and the expression of 25 kDa recombinant CMV coat protein was confirmed in 12 per cent sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS- PAGE). Tris- NaCl buffer of pH 8.0 was selected for solubilising the recombinant protein using ExPASy - protein translation tool. The recombinant protein was further purified through Nitrilotriacetic acid column purification, in which the 6X histidine tagged recombinant protein was bound with agarose coated nickel beads. Buffers containing imidazole were used for the elution of histidine tagged recombinant protein, since imidazole competes with histidine for the binding site in nickel beads. Each fraction viz., cell pellet, supernatant, flow through, wash and elution were collected and later detected for protein using SDS- PAGE. Absence of 25 kDa protein in cell pellet indicated that the recombinant coat protein completely soluble in Tris- NaCl buffer (pH 8.0). Confirmation of recombinant coat protein was carried out through DAC- ELISA and western blotting using commercially available polyclonal CMV antiserum (1: 2000; NRCB, Trichy). The recombinant coat protein developed through this study could be utilised for large scale production of antiserum for immunodetection of CMV.Item Cloning and expression of coat protein gene of sweet potato leaf curl virus (splcv)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Sruthy, G S; Makeshkumar, T